Project Summary Respiratory tract infections (RTI) are the 4th-leading cause of the morbidity and mortality in the United States and seventh worldwide. There are between 10 and 20 well-known bacterial and viral pathogens (not including numerous subtypes and genotypes) which are considered as the main causes of RTI. Current in-vitro diagnostic (IVD) tests for RTI pathogens are based on PCR (RT-PCR) amplification of photogenic nucleic acids (NA). Detection of the amplified target genes can occur in real-time regime (real-time PCR) that provides fast turnaround time but can usually identify not more than five pathogens or pathogen genotypes per assay. On the other hand, employing post-amplification techniques such as hybridization with solid-phase spatially separated oligonucleotide probes (microarrays) can significantly enlarge the number of pathogens to be detected, but requires ?open-tube? manipulation with PCR amplification products, such as the transfer of the PCR products to microarray chamber and microarray washings. These open-tube procedures greatly complicate assay?s protocol, extend analysis time, and increase risks of sample cross-contamination. Here, the PI proposes to develop and demonstrate a proof-of-concept assay for fast (<30 min) identification and discrimination of 10 common RTI pathogens using an innovative multiplexed RT-PCR platform, named the Donut PCR. The platform includes a disposable microfluidic reaction chamber where amplification occurs due to convection-based thermocycling. The chamber equipped with an embedded label-free microarray allowing real-time detection of amplification products without opening the reaction chamber. To run the assay, the team has developed a complementary PCR instrument which is characterized by the easiness of operation, small footprint and low energy consumption (functional prototype complete). The team anticipates that the demonstrated assay will constitute a solid basis for further development of cost-effective, rapid IVD systems capable of fast identification of a large variety of genetic targets.